title: A duplex real-time RT-PCR assay for profiling inhibitors of four dengue serotypes.
abstract: We have developed a duplex real-time RT-PCR assay for profiling antiviral inhibitors of four dengue virus (DENV) serotypes. In this assay, the primers and the probe for amplifying DENV were designed in the conserved regions of the genome after aligned more than 300 nucleotide sequences of four dengue serotypes deposited in the GeneBank. To discriminate the antiviral activity from the cytotoxicity of compounds, a housekeeping gene of the Vero cells, β-actin, was used to design the primers and the probe for the second set of PCR as an internal control, which is used to normalize the RNA levels of dengue-specific PCR due to the cellular toxicity of test compounds. For compound profiling, the duplex PCR is performed using LightCycler(®) in a single tube to simultaneously amplify both the dengue target gene and the Vero cell housekeeping gene from the compound-treated Vero cell lysates. This assay was validated against a panel of reference compounds. The results show that the universal primers and probe in this duplex RT-PCR assay can efficiently amplify all four dengue serotypes and that the PCR efficiency for both the dengue target gene and the Vero cells β-actin gene is 100%.
authors.affiliation: Janssen Infectious Diseases-Diagnostics BVBA, Johnson & Johnson Corporation, Beerse, Belgium.
authors.forename: Edwin Yunhao
authors.initials: EY
authors.lastname: Gong
authors.forename: Alexandra
authors.initials: A
authors.lastname: Smets
authors.forename: Nick
authors.initials: N
authors.lastname: Verheyen
authors.forename: Marleen
authors.initials: M
authors.lastname: Clynhens
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Gustin
authors.forename: Pedro
authors.initials: P
authors.lastname: Lory
authors.forename: Guenter
authors.initials: G
authors.lastname: Kraus
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002522:Chlorocebus aethiops; D003715:Dengue / Q000145:classification / Q000188:drug therapy* / Q000235:genetics*; D006801:Humans; D020128:Inhibitory Concentration 50; D008826:Microbial Sensitivity Tests / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012703:Serotyping; D014709:Vero Cells
chemical_list: D000998:Antiviral Agents